2 Enzymatic Modification of DNA and RNA
33
purified by using a chaotropic buffer (Nal)/silica-gel spin column system
(e. g. Qiagen) to remove all traces of buffer.
Subprotocol 2
Exonucleases, Other Nucleases and Ligases
In contrast to restriction enzymes, nucleases cleave DNA and RNA at nonspecific sites. Substrate for nucleases could be double stranded or single
stranded polynucleotides. They are degraded either from the termini ( exonucleases) or from within the sequence (endonucleases). In this section,
three examples are given for enzymes that are of some use in molecular
biology.
Deoxyribonudease I (DNase I)
This enzyme is isolated from bovine pancreas. DNase I is an endonuclease
that digests both double-stranded and single-stranded DNA. It displays two
modes of action depending on the ions added to the reaction.
• In the presence ofMg++ ions, each DNA strand is attacked independently
with cleavage sites distributed randomly. In this way, the enzyme is used
for introducing random nicks (i. e. discontinuities in one strand of a double-stranded molecule) into intact DNA prior to radiolabeling (nick
translation).
• In the presence of Mn++ ions, DNase I cleaves the complementary
strands of double stranded DNA at approximately the same sites to yield
very short, mostly blunt-ended fragments.
Nudease Sl
Nuclease SI is isolated from Aspergillus oryzae. It specifically degrades single-stranded DNA or RNA, whereas double-stranded molecules are resistant to cleavage. If the amount of enzyme in a reaction is increased, doublestranded DNA is nicked. This enzyme is used to remove single-stranded
tails or loops from DNA fragments, and to analyze the structure of
DNA: RNA hybrids.
33
purified by using a chaotropic buffer (Nal)/silica-gel spin column system
(e. g. Qiagen) to remove all traces of buffer.
Subprotocol 2
Exonucleases, Other Nucleases and Ligases
In contrast to restriction enzymes, nucleases cleave DNA and RNA at nonspecific sites. Substrate for nucleases could be double stranded or single
stranded polynucleotides. They are degraded either from the termini ( exonucleases) or from within the sequence (endonucleases). In this section,
three examples are given for enzymes that are of some use in molecular
biology.
Deoxyribonudease I (DNase I)
This enzyme is isolated from bovine pancreas. DNase I is an endonuclease
that digests both double-stranded and single-stranded DNA. It displays two
modes of action depending on the ions added to the reaction.
• In the presence ofMg++ ions, each DNA strand is attacked independently
with cleavage sites distributed randomly. In this way, the enzyme is used
for introducing random nicks (i. e. discontinuities in one strand of a double-stranded molecule) into intact DNA prior to radiolabeling (nick
translation).
• In the presence of Mn++ ions, DNase I cleaves the complementary
strands of double stranded DNA at approximately the same sites to yield
very short, mostly blunt-ended fragments.
Nudease Sl
Nuclease SI is isolated from Aspergillus oryzae. It specifically degrades single-stranded DNA or RNA, whereas double-stranded molecules are resistant to cleavage. If the amount of enzyme in a reaction is increased, doublestranded DNA is nicked. This enzyme is used to remove single-stranded
tails or loops from DNA fragments, and to analyze the structure of
DNA: RNA hybrids.